Bacillus cereus C1-3 capable of resisting tobacco mosaic virus, application of bacillus cereus C1-3 and tobacco planting method

Abstract

The invention discloses a bacillus cereus C1-3 for resisting tobacco mosaic virus and application thereof, relates to the technical field of microbial control, and comprises the bacillus cereus C1-3 for resisting tobacco mosaic virus, the bacillus cereus C1-3 is preserved in China Center for Type Culture Collection on July 18, 2024, the preservation number is CCTCC NO: M 20241595, the preservation number is CCTCC NO: M 20241595, and the preservation number is CCTCC NO: M 20241595. The preservation address is Wuhan University Campus, Wuchang District, Wuchang District, Hubei Province, Eight Road 299; the invention discloses application of bacillus cereus C1-3 in prevention and treatment of tobacco mosaic disease. The effective component of the preparation for resisting the tobacco mosaic virus comprises the bacillus cereus C1-3. The bacterial strain has the beneficial effects that the bacterial strain has a remarkable inhibition effect on tobacco common mosaic virus, can induce tobacco to generate disease resistance, achieves a better antiviral effect, and can be applied to development of mosaic virus resistant biopesticides.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial control, relates to a microbial control technology for tobacco mosaic disease, and specifically relates to a Bacillus cereus C1-3 resistant to tobacco mosaic virus and an application thereof and a tobacco planting method. Background Art

[0002] Tobacco mosaic virus (TMV) is an important tobacco disease. Tobacco virus diseases are common and widely distributed in tobacco-growing areas around the world, causing serious harm and seriously affecting tobacco yield and quality.

[0003] The current strategy for TMV prevention and control is mainly prevention, mainly because tobacco has a short growth cycle and TMV spreads quickly. Once the disease begins, it is difficult to prevent and control it in time. In response to this situation, the current prevention and control methods are mainly divided into three categories: agricultural prevention, chemical prevention and biological prevention. Biological pesticides, also known as bio-source pesticides, refer to some natural substances produced by organisms or biological metabolism that are directly used as pesticides or preparations to combat pests and diseases. This biological-based prevention and control method provides a more environmentally friendly and sustainable solution. Biopesticides can be divided into microbial pesticides, botanical pesticides, biochemical pesticides, etc. With the advancement and development of agricultural science and technology, green biopesticides, especially microbial pesticides, have gradually attracted attention. Microbial pesticides mainly include agents made from living organisms or metabolites such as fungi, bacteria, actinomycetes, and viruses. Among them, bacterial antagonists are widely used in the biological control of plant diseases. Bacillus is a Gram-positive bacterium that mostly lives a saprophytic life. The bacteria have endogenous spores, strong resistance to stress, fast reproduction, and simple nutritional requirements. Some species can secrete and produce more than a dozen active substances such as extracellular proteins, bacitracin, macrolides, cyclic peptides, and phage-like particles. Some Bacillus can also produce a variety of enzymes and polysaccharides, which have the function of promoting the growth of other organisms and stimulating their immune diseases. Studies have revealed the potential of Bacillus in resisting tobacco mosaic virus (TMV), such as Bacillus subtilis SN-02, which can produce a surfactant with antiviral properties. In addition, the metabolites of Bacillus amyloliquefaciens Ba33 showed an inhibitory effect on TMV in in vitro experiments and were able to promote the growth of tobacco plants; Bacillus subtilis Tpb55, which can not only effectively reduce the activity of TMV, but also stimulate the tobacco plant's own defense mechanism and enhance its resistance to TMV. As a microbial group with rich antiviral resources, Bacillus has great development and application prospects in biological control. Bacillusspp.) plays a functional role in inhibiting plant diseases due to its advantages such as being green and environmentally friendly, good stability, rapid growth, broad antibacterial spectrum, strong stress resistance and high biosafety. It is one of the most promising and commercially valuable biocontrol bacteria. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art. In order to reduce the use of chemical agents and the harm to the environment, and at the same time overcome the disadvantage that the control effect of biological agents is unstable when used in the field, the present invention provides a non-toxic, non-pathogenic, safe for humans and animals, environmentally friendly, strong in stress resistance, and stable in antibacterial activity for inhibiting plant viruses. Bacillus cereus C1-3 provides a new safe, efficient, and environmentally friendly biological pesticide for plant virus control, which helps promote the green and sustainable development of agriculture.

[0005] The technical solution of the present invention is as follows: The first aspect of the present invention provides a tobacco mosaic virus resistant Bacillus cereus C1-3, the Bacillus cereus ( Bacillus cereus ) C1-3 was deposited in the China Center for Type Culture Collection on July 18, 2024, with the deposit number CCTCC NO: M 20241595. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] Preferably, the Bacillus cereus C1-3 forms round or approximately round, soft, non-pigmented, slightly shiny white colonies on a beef extract peptone medium plate at 37° C. for 24 hours.

[0007] The second aspect of the present invention provides the use of the Bacillus cereus C1-3 in preventing and treating tobacco mosaic disease.

[0008] The third aspect of the present invention provides a method for controlling tobacco mosaic disease using the above-mentioned Bacillus cereus C1-3, the method comprising the following steps: inoculating Bacillus cereus C1-3 on a liquid culture medium and culturing the culture medium to obtain a fermentation liquid; The fermentation liquid is sprayed on tobacco leaves.

[0009] Preferably, the tobacco leaves are three-grown tobacco leaves.

[0010] Preferably, the liquid culture medium is beef extract peptone liquid culture medium.

[0011] Preferably, the culture conditions are: constant temperature shaking culture at 37°C and 200 r / min for 1 day to obtain Bacillus cereus C1-3 fermentation liquid, which is stored at 4°C for later use.

[0012] The fourth aspect of the present invention provides an anti-tobacco mosaic virus preparation, the active ingredient of which comprises the Bacillus cereus C1-3.

[0013] The fifth aspect of the present invention provides a pharmaceutical composition for resisting tobacco mosaic virus, wherein the active ingredient of the pharmaceutical composition comprises the Bacillus cereus C1-3.

[0014] A sixth aspect of the present invention provides a tobacco planting method, comprising the step of spraying the biological agent or the pharmaceutical composition on tobacco leaves.

[0015] The present invention has at least one of the following beneficial effects: The present invention isolates a strain of Bacillus cereus C1-3, which has a significant inhibitory effect on tobacco mosaic virus. First, in the three-year tobacco spot test, the inhibition rate of TMV is high, and the inhibition rate of Bacillus cereus C1-3 fermentation liquid on TMV passivation can reach 82.12%. Further exploration of the inhibitory effect of Bacillus cereus C1-3 fermentation liquid on TMV shows that Bacillus cereus C1-3 can induce tobacco to produce disease resistance from the changes in the virus expression amount and defense enzymes in K326 tobacco. After inoculation with TMV, the resistance generated in the body makes it impossible for the tobacco-infected virus to reproduce in large quantities in the body, thereby achieving a good antiviral effect. The application of Bacillus cereus C1-3 in the development of anti-mosaic virus biopesticides can develop new, efficient and safe microbial preparations to control plant viral diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The culture morphological characteristics and Gram staining observation results of strain C1-3 in Example 1 of the present invention are shown; Figure 2 is the phylogenetic tree of strain C1-3 in Example 1 of the present invention; Figure 3 This is a photo of tobacco leaves treated with the C1-3 fermentation liquid treatment group and the CK control group in Example 2 of the present invention; Figure 4 This is a graph showing changes in TMV expression levels in the C1-3 fermentation broth treatment group and the CK clear water control group in Example 2 of the present invention; Figure 5 This is a graph showing the results of measuring the defense enzyme activity of the C1-3 fermentation broth treatment group and the CK clear water control group in Example 2 of the present invention. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0018] The present invention is further described in detail below with specific examples, but the present invention is not limited to the following specific examples.

[0019] Example 1 Screening of Bacillus cereus C1-3 1. Isolation and purification of strains Strain C1-3 was isolated from the perennial rotation tobacco soil of the tobacco planting base in Tanxi Township, Lichuan County, Fuzhou City, Jiangxi Province by pure culture. The specific method is to use the soil collected from the tobacco planting base in Tanxi Township, Lichuan County, Fuzhou City, Jiangxi Province during the tobacco harvest period in July 2020 and June 2021 for tobacco cultivation. When the tobacco grows to 6-7 leaves, the root soil is taken for separation and purification of soil microorganisms. After dilution according to the conventional separation method, it is spread on beef extract peptone medium, potato agar medium (PDA), and Gao's No. 1 medium, and cultured in a constant temperature incubator for 2-5 days. Among them, Gao's No. 1 medium and PDA medium are cultured in a 28°C incubator, and beef extract peptone medium is cultured in a 37°C incubator. Pick a single colony for plate streaking purification, and the purified colonies are used for TMV passivation test and plant disease resistance induction test, and the effective bacteria are sent for testing and preservation.

[0020] Results: The strain C1-3, which had a high inactivation effect on TMV and could induce disease resistance in plants, was successfully isolated from beef extract peptone culture medium.

[0021] Formula of beef extract peptone solid culture medium: peptone 10g, sodium chloride 5g, beef extract 3g, agar 15-20g. Dissolve in 400ml water first, then add water to 1000ml, adjust pH to 7.2±0.3, and sterilize at 121℃ for 20min.

[0022] Gao's medium No. 1 and PDA medium are commercially available culture media.

[0023] 2. Strain identification The strain C1-3 was identified by morphological characteristics and 16SrDNA sequence analysis, as follows: 2.1. Culture morphological characteristics of strains Morphological observation and culture characteristics: The strain was streaked and inoculated on beef extract peptone medium, and after culturing at 37°C for 1 day, its morphological characteristics such as colony size, color, and morphology were observed under a microscope, and then a single colony was picked for staining observation.

[0024] Gram staining: First, fix the smear, stain it with crystal violet for 1 minute, wash it with sterile water, stain it with iodine solution for 1 minute, wash it with sterile water, decolorize it with 95% ethanol, rinse it and stain it with safranin solution for 10 seconds. The staining result shows purple, which is Gram-positive bacteria.

[0025] The results are as follows Figure 1 As shown, the Bacillus cereus C1-3 forms round or approximately round, soft, non-pigmented, slightly shiny white colonies on a beef extract peptone medium plate at 37° C. for 24 hours; the bacterial morphology is Gram-positive and rod-shaped under microscope observation.

[0026] 2.2. 16S rDNA sequence analysis and identification The DNA of the strain was extracted using the Novozymes bacterial DNA extraction kit. After extracting the total genomic DNA of the strain, universal primers for the 16 S rDNA gene were designed. The sequences of the amplification primers were: Forward 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; Reverse 1492R: 5′-GGTTACCTTGTTACGACTT-3′; The length of the amplified fragment was approximately 1437 bp.

[0027] The extracted total DNA was used as a PCR template, and the PCR products were analyzed by electrophoresis using 1% agarose gel, and the corresponding results were observed using a gel imager. The SanPrep column DNA gel recovery kit from Beijing Qingke Biotechnology Co., Ltd. was used. The recovered PCR products were sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared for homology in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the phylogenetic tree of strain C1-3 was constructed using MEGA11 software, as shown in the figure. Figure 2 shown.

[0028] The 16S rDNA sequencing result of Bacillus cereus C1-3 is shown in SEQ ID NO: 1:

[0029] The strain was deposited in the China Center for Type Culture Collection on July 18, 2024, and its classification name is Bacillus cereus ( Bacillus cereus ) C1-3, the deposit number is CCTCC NO: M 20241595, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0030] Example 2 Detection of antiviral activity of Bacillus cereus C1-3 fermentation broth 1. Preparation of Bacillus cereus C1-3 fermentation broth The Bacillus cereus C1-3 screened in Example 1 was inoculated into a beef extract peptone liquid culture medium (preparation method: 3 g beef extract, 10 g peptone, 5 g NaCl, add water to 1000 mL, adjust pH 7.0-7.2), and cultured at 37°C, 200 r / min for 1 day with constant temperature shaking to obtain a Bacillus cereus C1-3 fermentation broth, which was stored at 4°C for later use.

[0031] 2. The effect of Bacillus cereus C1-3 fermentation liquid on TMV control in tobacco by dead spot method Select healthy 8-leaf Nicotiana tabacum var samsun NN leaves with uniform size and symmetrical midribs.

[0032] Take ordinary tobacco leaves infected with TMV and showing severe symptoms of tobacco mosaic disease, grind and mix them with inoculated PBS buffer (10mM phosphate buffer, pH7.4) at a ratio of 1:10 (w / v) to serve as TMV virus inoculation solution.

[0033] C1-3 fermentation liquid treatment group: Take the Bacillus cereus C1-3 fermentation liquid obtained above and mix it with TMV virus inoculation liquid at a ratio of 1:1 (v / v). Place the mixture at 25℃ and let it stand for 30 minutes. Then dip it into the inoculation liquid and rub it on the right half of the leaf of the selected 8-leaf Sansheng tobacco.

[0034] CK control group: Mix the above PBS buffer solution with TMV virus inoculation solution at a ratio of 1:1 (v / v). After half an hour, rub and inoculate the left half of the leaf as the control group.

[0035] Six leaves were inoculated in each treatment, with three replicates.

[0036] After obvious dead spots appear, record the number of dead spots, take the average value, and calculate the inhibition rate according to the following formula: Inhibition rate (%) = [1-(number of treated lesions / number of control lesions)] x 100%.

[0037] The number of treated necrotic spots is the average number of necrotic spots in the C1-3 fermentation liquid treatment group (right half of the leaf), and the number of control necrotic spots is the average number of necrotic spots in the CK control group (left half of the leaf).

[0038] 3. Mechanism of Anti-TMV Activity of Bacillus cereus C1-3 Fermentation Broth on Tobacco We selected healthy and uniformly growing common tobacco K326 at the 6-8 leaf stage and set different treatments: CK: TMV virus inoculum was inoculated 24 h after spraying with clean water; Group C1-3: The fermentation liquid of Bacillus cereus C1-3 was sprayed and then inoculated with the TMV virus inoculum obtained above 24 hours later.

[0039] Leaves from the same part of tobacco in each group were taken at 1, 3, 5, 7, and 9 days after inoculation, and 3 plants were randomly selected for each treatment, repeated 3 times. The leaves were collected and quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator to determine the activity of related defense enzymes and the expression of viruses by qRT-PCR.

[0040] 3.1. qRT-PCR measurement of viral expression The RNA of the leaves collected above was extracted using the Trizol method. 50-100 mg of fresh plant tissue was minced as much as possible, and then 1 mL of Total RNA Extraction Reagent was added to mix well. The homogenized sample was shaken vigorously and placed at room temperature for 5 minutes to completely dissociate the ribosomes. Centrifuge at 4°C, 12, 000 rpm for 10 min, and the supernatant was collected. 0.2 mL of chloroform was added to the above lysate. The centrifuge tube was tightly capped, and the tube was shaken vigorously for 15 sec. The tube was placed at room temperature for 2-3 min. Centrifuge at 4°C, 12,000 rpm for 10-15 min. The upper aqueous phase was carefully pipetted into a new centrifuge tube, and an equal volume of isopropanol was added. Inverted to mix well and placed at room temperature for 10 min. Centrifuge at 4°C, 12, 000 rpm for 10 min. The supernatant was carefully discarded and 1 mL of 75% ethanol was added. Vortex to wash thoroughly, and flick the bottom of the tube to suspend the precipitate. Centrifuge at 4℃, 12,000 rpm for 5 min and discard the supernatant. Air dry at room temperature for 5-10 min. Add 30-100 μL RNase-free water to dissolve the RNA. After it is completely dissolved, take a small amount for testing and store the rest of the solution at -80℃.

[0041] The nucleic acid concentration of the extracted total RNA was determined using a Nanodrop ultra-micro spectrophotometer. After the determination, tobacco cDNA was synthesized and amplified. Real-time fluorescence quantitative PCR was used to determine the TMV content in tobacco. β-Actin in common tobacco K326 samples treated at different time periods was used as the internal reference gene, and TMV-CP in tobacco treated with the fermentation broth of each strain was used as the target gene content to evaluate the effect of the fermentation broth of each strain on tobacco resistance. The relative expression level of the TMV-CP gene under each treatment at different time periods was calculated based on 2-ΔΔCt.

[0042] 3.2. Determination of defense enzyme activity The peroxidase (POD) activity was determined by the guaiacol method. The reaction system was 2.9 mL 0.05 mol / L PBS buffer (pH 5.5), 1.0 mL 2% H2O2, 1.0 mL 0.05 mol / L guaiacol, and 0.1 mL enzyme solution. The mixture was kept in a 37°C water bath for 5 min, and PBS was used as a blank control instead of enzyme solution. After mixing, the timer was immediately started, and the absorbance change within 3 min was measured at 470 nm. A change of 0.01 per minute was considered an enzyme activity unit U / (g·min).

[0043] The activity of superoxide dismutase (SOD) was determined using a superoxide dismutase activity detection kit. 0.1 g of tobacco leaves were added with 1 mL of extract and homogenized in an ice bath. The mixture was centrifuged at 8,000 g and 4 °C for 10 min, and the supernatant was taken as the crude enzyme solution, which was placed on ice for testing. The crude enzyme solution was added to the reagent and mixed well. The premixed solution without the sample was the blank control group. After being incubated in a 37 °C water bath for 30 min, the solution was placed in a 1 mL glass cuvette and the absorbance at 560 nm was measured.

[0044] Phenylalanine ammonia lyase (PAL) activity was determined using a phenylalanine ammonia lyase detection kit. 0.1 g of tobacco leaves were added with 1 mL of extract and homogenized in an ice bath; centrifuged at 8,000 g, 4 °C for 10 min, and the supernatant was taken as the crude enzyme solution, which was placed on ice for testing. The crude enzyme solution was added to the reagent and mixed well. The premix without sample was used as the blank control group. After standing for 10 min, the absorbance of the measuring tube was recorded at 290 nm.

[0045] 4. Test Results 4.1 Results of the test on the control effect of Bacillus cereus C1-3 fermentation liquid on tobacco by the dead spot method After calculation, the inhibition rate was 82.12%, indicating that the fermentation liquid of Bacillus cereus C1-3 in this example had a preventive and therapeutic effect on tobacco TMV.

[0046] Figure 3 The pictures show the tobacco leaves after being treated with the C1-3 fermentation liquid treatment group (right half of the leaf) and the CK water control group (left half of the leaf). Figure 4 The left half of the leaves showed obvious yellow-green mottled spots, while the right half of the leaves did not show obvious yellow-green mottled spots, indicating that the C1-3 fermentation broth had an anti-TMV effect.

[0047] Figure 4 The figure shows the changes in TMV expression in the C1-3 fermentation broth treatment group and the CK water control group. Figure 4 It can be seen that on the 1st, 3rd, 7th and 9th days, the expression levels of TMV in the C1-3 fermentation liquid treatment group were significantly lower than those in the CK water control group, indicating that the C1-3 fermentation liquid can effectively inhibit the proliferation of TMV in tobacco plants.

[0048] Figure 5 The results show the determination of defense enzyme activity. The activities of POD, PAL, and SOD in tobacco treated with the fermentation liquid of C1-3 strains were increased to varying degrees compared with the CK group. The results showed that POD activity reached a peak on the third day; PAL activity reached a peak on the third day; and SOD activity reached a peak on the ninth day.

[0049] In summary, it can be seen from the changes in virus expression and defense enzymes in K326 tobacco that Bacillus cereus C1-3 can induce disease resistance in tobacco. After inoculation with TMV, the resistance produced in its body prevents the tobacco-infected virus from multiplying in large quantities in its body, thereby achieving a better antiviral effect.

[0050] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A tobacco mosaic virus resistant Bacillus cereus C1-3, characterized in that: The Bacillus cereus ( Bacillus cereus ) C1-3 was deposited in the China Center for Type Culture Collection on July 18, 2024, with the deposit number CCTCC NO: M 20241595. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The Bacillus cereus C1-3 according to claim 1, characterized in that The Bacillus cereus C1-3 forms round or approximately round, soft, non-pigmented, slightly shiny white colonies on a beef extract peptone medium plate at 37° C. for 24 hours.

3. Use of Bacillus cereus C1-3 as described in any one of claims 1 to 2 in preventing and treating tobacco mosaic disease.

4. A method for preventing and treating tobacco mosaic disease using Bacillus cereus C1-3 as described in any one of claims 1 to 2, characterized in that: The method comprises the following steps: inoculating Bacillus cereus C1-3 on a liquid culture medium and culturing the culture medium to obtain a fermentation liquid; The fermentation liquid is sprayed on tobacco leaves.

5. The method for preventing and treating tobacco mosaic disease according to claim 4, characterized in that: The tobacco leaves are three-year-old tobacco leaves.

6. The method for preventing and controlling tobacco mosaic disease according to claim 4, characterized in that: The liquid culture medium is beef extract peptone liquid culture medium.

7. The method for preventing and controlling tobacco mosaic disease according to claim 4, characterized in that: The culture conditions are: constant temperature shaking culture at 37° C. and 200 r / min for 1 day to obtain Bacillus cereus C1-3 fermentation liquid, which is stored at 4° C. for later use.

8. A biological agent against tobacco mosaic virus, characterized in that: The active ingredient comprises the Bacillus cereus C1-3 described in any one of claims 1 to 2.

9. A pharmaceutical composition for resisting tobacco mosaic virus, characterized in that: The active ingredient comprises the Bacillus cereus C1-3 described in any one of claims 1 to 2.

10. A tobacco planting method, characterized in that: The method comprises the step of spraying the biological agent according to claim 8 or the pharmaceutical composition according to claim 9 onto tobacco leaves.

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